Despite considerable advances in our understanding of borrelial differential gene expression, the molecular interactions and regulatory events that enable Borrelia burgdorferi to cycle between its arthropod vector and mammal host are not well understood. The studies presented in this proposal are predicated on the notion that a more thorough analysis of Bb within infected ticks will provide valuable insight into the virulence-related processes involved in spirochete transmission. Our preliminary studies using a GFP+ strain 297 isolate challenge the conventional thinking of how Bb migrate out of the nymphal midgut during feeding. During the bloodmeal, Bb replicate within the tick midgut as non-motile networks, gaining access to the basement membrane by taking advantage of the weakness created by midgut remodeling. Bb then undergo a dramatic transition from the non-motile networks to individual motile organisms that are able to penetrate through the MG epithelium into the hemocoel, migrate to the salivary glands, and ultimately, infect mammalian host tissues. In this application, we propose using a combination of molecular, genetic and microscopic imaging methodologies to more precisely delineate the interactions that occur between the Lyme disease spirochete and its arthropod vector throughout the feeding process. By refining our understanding of the interplay between spirochetal and tick factors, we will further our long-term objective to identify borrelial, as well as ixodid, gene products and regulatory pathways that promote spirochete dissemination within both ticks and mammals. By identifying key components acting at the pathogen-vector interface, we will be setting the stage for the development of novel therapeutic strategies, including vaccine candidates capable of blocking transmission to, as well as dissemination within, the human host. PUBLIC HEALTH RELEVANCE: Borrelia burgdorferi (Bb), the causative agent of Lyme disease (LD), is maintained within nature by an enzootic life cycle that involves an Ixodes tick vector and a mammalian host, typically wild rodents. The studies presented in this proposal are predicated on the notion that a more thorough analysis of spirochetes within ticks will provide valuable insight into how Bb is transmitted to its reservoir host and humans. By refining our understanding of the interplay between Bb and the tick midgut epithelium, we will further our long-term objective to identify borrelial, as well as tick, gene products and regulatory pathways that promote maintenance of Bb within its tick-mouse infectious cycle.